Mechanisms of Leukocyte Distribution During Sepsis

Ploppa et al. Critical Care 2010, 14:R201 http://ccforum.com/content/14/6/R201

RESEARCH Open Access Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury Annette Ploppa1, Volker Schmidt1, Andreas Hientz2, Joerg Reutershan1, Helene A Haeberle1, Boris Nohé1*

Abstract Introduction: This study was carried out to determine whether interactions of cell activation, shear stress and platelets at sites of endothelial injury explain the paradoxical maldistribution of activated leukocytes during sepsis away from local sites of infection towards disseminated leukocyte accumulation at remote sites. Methods: Human umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were activated with lipopolysaccharide at 100 and 10 ng/ml to achieve adhesion molecule patterns as have been reported from the hyper- and hypo-inflammatory stage of sepsis. To examine effects of leukocyte activation on leukocyte-endothelial interactions, activated HUVEC were perfused with activated and non-activated neutrophils in a parallel plate flow chamber. Adhesion molecule expression and function were assessed by flow cytometry and blocking antibodies. In a subset of experiments the sub-endothelial matrix was exposed and covered with platelets to account for the effects of endothelial injury. To investigate interactions of these effects with flow, all experiments were done at various shear stress levels (3 to 0.25 dyne/cm2). Leukocyte-endothelial interactions were analyzed by videomicroscopy and analysis of covariance. Results: Activation of neutrophils rendered adhesion increasingly dependent on shear stress reduction. At normal shear stress, shedding of L-selectin decreased adhesion by 56%. Increased rolling fractions of activated PMN at low shear stress revealed impaired integrin affinity despite numerical up-regulation of CD11b. On sub-maximally activated, intact HUVEC shear stress became the prevailing determinant of adhesion. Presence of a platelet-covered injury with high surface density of P-selectin was the strongest variable for adhesion. When compared to maximally activated HUVEC, platelets increased neutrophil adhesion by 2.7-fold. At sub-maximal activation a 10-fold increase was observed (P < 0.05 for all). Conclusions: L-selectin shedding and integrin dysfunction render leukocyte adhesion increasingly susceptible to shear stress and alternative adhesion receptors. In combination, these effects inhibit recruitment to normally perfused sites with intact endothelium and favor maldistribution towards sites with compromised perfusion or endothelial injury.

Introduction upregulation of endothelial adhesion molecules in Directing leukocytes to local sites of infection is a cru- inflamed tissue, resulting in a targeted accumulation of cial part of the innate immune response. While intravas- leukocytes at the site of infection [1]. Initially, selectin- cular shear forces prevent relevant leukocyte adhesion in dependent interactions overcome postcapillary shear a healthy individual, increased concentrations of micro- stress, enabling capture and rolling of leukocytes on the bial toxins and pro-inflammatory mediators induce activated endothelium. Selectin-interactions and local chemokines then activate leukocyte integrins such as * Correspondence: [email protected] lymphocyte function antigen-1 (LFA-1, CD11a/CD18) 1Department of Anesthesiology and Intensive Care Medicine, Tuebingen and macrophage antigen-1 (MAC-1, CD11b/CD18). University Hospital, Eberhard-Karls University, Hoppe-Seyler-Str. 3, Tuebingen, Local activation of integrins favours interactions with 72076, Germany Full list of author information is available at the end of the article endothelial counter-receptors, such as intercellular

© 2010 Ploppa et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ploppa et al. Critical Care 2010, 14:R201 Page 2 of 13 http://ccforum.com/content/14/6/R201

adhesion molecule-1 (ICAM-1), resulting in firm Boehringer, Mannheim, Germany) and cultured in adhesion [1]. Endothelial Cell Growth Medium (EGM™,PromoCell, In contrast to local inflammation, systemic sepsis is Heidelberg, Germany) on collagen-coated rectangular characterized by profound leukocyte activation through- coverslips (Falcon Biocoat™, Becton Dickinson Labware, out the circulation [2,3]. Because organ damage is attenu- Bedford, MA, USA). Confluent HUVEC of the first pas- ated by inhibiting leukocyte-endothelial interactions, sage were used for the experiments. systemic leukocyte activation and disseminated leukocyte PMN were isolated by density gradient centrifugation adhesion are regarded essential for septic organ dysfunc- at 1,700 rpm on a discontinuous Percoll gradient with tion [4-7]. In the last few years this traditional assump- 63% and 72% Percoll in buffer (Percoll, 1.130 g/ml; tion has been challenged by the finding of an impaired Amersham Pharmacia Biotech, Uppsala, Sweden). The chemotaxis and decreased rather than increased leuko- bottom layer was collected and contaminating erythro- cyte recruitment to local sites of infection in septic indi- cytes were removed by hypotonic lysis in 10% NH4Cl on viduals despite persistent upregulation of leukocyte ice. After washing, the PMN pellet was resuspended in integrins [2,3,8-10]. Moreover, it has been recognized cold Medium 199 (Sigma, St. Louis, MO, USA) supple- that systemic hyper-inflammation often turns into hypo- mented with 50% fetal calf serum (Gibco, Mannheim, inflammation with immunosuppressive cytokine-profiles Germany) at 5 × 107/ml. To avoid assay related activa- such as increased ratios of interleukin (IL)-10 and tumor tion of PMN during rewarming, we reconstituted the necrosis factor (TNF)-a [11-13]. Similar to the phenom- PMN pellet to 106 PMN/ml just before the adhesion enon of endotoxin tolerance, endothelial sensitivity to assay in normoxic, room temperature Medium 199 only. microbial toxins becomes altered and endothelial cell Final rewarming to 37°C was achieved in the heatable adhesion molecule expression is impaired [14-17]. Para- flow chamber. doxically these changes do not seem to protect patients from the development of endothelial cell damage and Adhesion assay leukocyte-related organ dysfunction since they are most PMN adhesion to HUVEC was quantified in a parallel pronounced in those with poor prognosis [12,13]. To plate flow chamber with a laminar flow profile (Rey- provide more insight into themechanismsthatcontri- nolds number <1, Figure 1) at 37°C as previously bute to these apparently paradoxical findings, we investi- reported [18]. According to those shear forces that have gated the following questions in a flow chamber model been observed in postcapillary venules of normal and with lipopolysaccharide induced inflammation. septic individuals we varied shear stress from 3 to 0.25 First, does systemic leukocyte activation increase or dyne/cm2 [19-25]. impair leukocyte recruitment to activated endothelium PMN were perfused over HUVEC-containing cover- and what are the mechanisms during the different stages slips for 10 minutes under different conditions of LPS- of inflammation? Second, if targeted leukocyte recruit- activation. Thereafter, PMN-adhesion was determined ment to locally activated endothelium is impaired, are from 10 s video recordings of five different fields of there mechanisms that favour disseminated leukocyte view by phase contrast microscopy (20× objective; accumulation at the same time? Third, given that later DMIRB, Leica, Bensheim, Germany). PMN were defined sepsis is characterized by immunosuppression, endothe- as rolling when traveling below 50% of the velocity of lial cell damage and organ dysfunction, are there free flowing PMN in close proximity to the endothelium mechanisms, independent of the physiological immune at the given shear stress [26]. A PMN, moving less than response, that gain a leading role for the distribution of one cell diameter in 10 s was defined to be firmly adher- leukocyte accumulation? ent. To exclude sedimentation artefacts, we exposed the adherent PMN, stepwise, up to 32 dyne/cm2 after the Materials and methods end of the adhesion experiment and measured cell Endothelial cell culture and leukocyte separation detachment. Under this exposure >70% of the adherent In compliance with the Helsinki Declaration on experi- PMN remained bound. As a measure for adhesion effi- mental research on humans and after obtaining ethical ciency [27,28], the rolling fraction was calculated as: committee approval (local ethics committee, University *(No. of rolling cells) × 100)/(No. of rolling cells + No. of of Tuebingen, reference numbers 315/99 and 69/2003- firmly adherent cells). Mean rolling velocities were A) and informed consent, human umbilical venous determined from more than 25 individual velocity pro- endothelial cells (HUVEC) and polymorphonuclear neu- files for each experimental condition as derived from trophils (PMN) were derived from human umbilical customized software for image recognition (CellTracker, veins and citrated blood samples from healthy volun- C. Zanke, University of Tuebingen, Germany). teers as previously described [18]. HUVEC were har- Selectin function was determined at 2 dyne/cm2 in vested by collagenase treatment (collagenase A 0.1%, presence of functional blocking monoclonal antibodies Ploppa et al. Critical Care 2010, 14:R201 Page 3 of 13 http://ccforum.com/content/14/6/R201

Figure 1 Parallel plate flow chamber. The flow chamber consisted of a heatable metal case (1). The silicone-sealed coverslips (2) were placed in the middle. Using a transparent cover block (3) with a flow channel (4) and a scaled metal ring (5), the chamber could be closed to a defined height leaving an inner chamber with a defined height of 0.2 mm. The tubing of the cell suspension was connected by a needle (6) to the inlet and outlet port of the transparent cover block (4). Temperature was controlled by temperature measurement within the metal case. Preliminary experiments showed that temperature of the metal block equaled with temperature of the perfusate within few seconds. For microscopy of the adhesion assay, the whole system was placed on an inverted phase-contrast microscope.

(mAb). PMN and HUVEC were incubated for 30 min- saturating amounts of fluorochrome conjugated mAb utes prior to the adhesion assay with mAb against against E-selectin, L-selectin (both from BD endothelial (E)-selectin (P2H3; Chemicon International, Biosciences), ICAM-1 (Immunotech, Marseille, France) Temecula, CA, USA), leukocyte (L)-selectin (DREG-56; and CD11b (Caltag, San Francisco, CA, USA). Matching BD Biosciences Pharmingen, San Jose, CA, USA), plate- isotype controls were used to define the setup of the let (P)-selectin (WASP12.2; Endogen, Woburn, MA, instrument. Unintended PMN-activation during cell USA) or a nonspecific antibody (HP6069; BD separation was ruled out by comparison of isolated Biosciences Pharmingen). PMN to leukocytes from whole blood.

Activation protocol modelling different stages of sepsis Activation protocol modelling endothelial injury By combining different conditions of neutrophil and Distinct from true endothelial activation, severe sepsis endothelial activation, we intended to mimic patterns of leads to endothelial cell injury which is likely to persist adhesion molecule expression as they have been observed even in the hypo-inflammatory stage [30,32] and results during local inflammation and different stages of sepsis- in platelet (PLT)-adhesion to the subendothelial matrix associated systemic hyper- or hypo-inflammation [33,34]. To account for PLT-PMN interactions under [1,2,8-10,29-31]. As detailed in Table 1, HUVEC were these conditions, we compared PMN-adhesion to acti- activated for four hours and PMN for 30 minutes with vated HUVEC with PMN-adhesion to PLT-treated, either 0 ng/ml, 10 ng/ml or 100 ng/ml LPS (026:B6 from injured HUVEC (Table 1) using a previously described Escherichia coli, Sigma), dissolved in Medium 199 sup- model for endothelial injury [33]. By pipetting medium at plemented with 20% fetal calf serum. high shear into the center of the coverslip an endothelial The changes in adhesion molecule expression were injury with exposure of the subendothelial matrix was determined by flow cytometry (FACSort™,Becton created. To allow for platelet-matrix interactions, the Dickinson, San Jose, CA, USA). Cells were gated using coverslips were perfused with citrated whole blood at forward and side scatter properties and staining with 20 dyne/cm2 for five minutes prior to the PMN adhesion Ploppa et al. Critical Care 2010, 14:R201 Page 4 of 13 http://ccforum.com/content/14/6/R201

Table 1 Description of the different groups and their activation protocol Group Activation Description HUVEC-/PMN- HUVEC 0 ng/ml LPS Control (non-inflamed tissue) + PMN 0 ng/ml LPS HUVEC++/PMN- HUVEC 100 ng/ml Maximal local inflammation LPS + PMN 0 ng/ml LPS HUVEC++/PMN ++ HUVEC 100 ng/ml Maximal systemical inflammation in the hyper-inflammatory stage of sepsis LPS + PMN 100 ng/ml LPS HUVEC+/PMN- HUVEC 10 ng/ml LPS Submaximal local inflammation + PMN 0 ng/ml LPS HUVEC+/PMN + HUVEC 10 ng/ml LPS Submaximal systemical inflammation in the hypo-inflammatory stage of sepsis + PMN 10 ng/ml LPS HUVEC++/PMN+ HUVEC 100 ng/ml Maximal systemical inflammation and endothelial damage in the hyper-inflammatory stage of sepsis +/PLT LPS + PMN 100 ng/ml LPS HUVEC+/PMN+/PLT HUVEC 10 ng/ml LPS Submaximal systemical inflammation and endothelial damage in the hypo-inflammatory stage of sepsis + PMN 10 ng/ml LPS HUVEC, human umbilical venous endothelial cells; PMN, polymorphonuclear neutrophils; PLT, platelets; LPS, lipopolysaccharide. assay. Since platelet-matrix interactions are much more ++/PMN++) resulted in maximal upregulation of E- shear-resistant than leukocyte-endothelial interactions, selectin, ICAM-1, CD11b and complete shedding of L- this resulted in dense platelet accumulation at the site of selectin, comparable to systemic hyper-inflammation injury without premature leukocyte adhesion. Before [10,29-31]. Similar to the hypo-inflammatory stage of sep- starting the PMN adhesion assay, the chamber was sis [2,3,10-17], submaximal activation with 10 ng/ml still cleared from remaining blood by a thorough rinse with upregulated CD11b and downregulated L-selectin on cell free medium. Then, the platelet-covered HUVEC PMN to the same degree as 100 ng/ml did, however, with- were perfused with the PMN suspension at 2 to 0.25 out having an effect on endothelial cell adhesion molecule dyne/cm2. expression (Figure 2).

Statistics Effects of cell activation, shear stress and their interplay All experiments were carried out in quadruplicate. The on PMN-HUVEC adhesion medians of fluorescence intensity (MFI) were calculated Normal shear stress of 2 to 3 dyne/cm2 prevented relevant from 5,000 single events by flow cytometry. An analysis of adhesion in non-activated HUVEC-/PMN-. As expected variance (ANOVA) was performed to determine whether in the model for local inflammation, maximal LPS- adhesion molecule expression was influenced by LPS acti- activation of HUVEC largely increased adhesion of non- vation. Using an analysis of covariance (ANCOVA) and activated PMN at 3 dyne/cm2 from 42 ± 17 (HUVEC-/ post hoc t-tests, we examined whether PMN activation PMN-) to 894 ± 93 cells/mm2 in HUVEC++/PMN- (P < (nominal effect), shear stress (continuous effect) or a com- 0.01, Figure 3a, b). In contrast, co-activation of PMN, in bination thereof influenced PMN adhesion. Effects of HUVEC++/PMN++, did not increase but decreased PMN platelets were analyzed accordingly (replacing PMN- adhesion by 56% when compared to HUVEC++/PMN- at activation by PLT-treatment). Effects of antibody blockade 3 dyne/cm2 (P < 0.01, Figure 3b). were examined by paired t-tests. Results of the adhesion At sub-maximal LPS-activation, activation of PMN in assays are presented as means ± SEM. A P-value <0.05 HUVEC+/PMN+ again decreased adhesion when com- after Bonferroni-Holm correction was considered signifi- pared to HUVEC+/PMN- (P < 0.01, Figure 3c). Despite cant. All analyses were performed using the statistical soft- persistent upregulation of CD11b this difference was ware JMP (SAS Institute Inc., Cary, NC, USA). most pronounced at low shear stresses where primary integrin-dependent adhesion becomes possible indepen- Results dent of selectin interactions [35]. When compared to non-activated controls (HUVEC-/ According to the effect of shear stress in general, PMN-), maximal LPS-activation with 100 ng/ml (HUVEC PMN adhesion increased with decreasing shear stress in Ploppa et al. Critical Care 2010, 14:R201 Page 5 of 13 http://ccforum.com/content/14/6/R201

Figure 2 Effects of different concentrations of LPS on the expression of adhesion molecules determined by flow cytometry. (a) ICAM-1, (b) E-selectin, (c) CD11b, (d) L-selectin. Induction of E-selectin and ICAM-1 expression on HUVEC required maximal activation with LPS, whereas the sub-maximal activation induced a shedding of L-Selectin and increase of CD 11b-expression on PMN (* P < 0.01 vs. 0 ng/ml; ANOVA of logarithms). all groups (Figure 3d-f). More importantly, analysis by HUVEC++/PMN++ reduced adhesion down to back- ANCOVA showed significant interaction between cell ground values observed in HUVEC-/PMN-. activation and shear stress. As soon as PMN were activated, adhesion became increasingly dependent on shear Effects of cell activation, shear stress and their interplay stress (P < 0.01, Figure 3e, f). on PMN-HUVEC-rolling interactions To determine whether a dissociation of quantitative and Relevance of selectin interactions for PMN adhesion to qualitative integrin upregulation contributed to the intact HUVEC decreased adhesion of LPS-activated PMN, rolling frac- Addition of selectin-blocking mAbs at 2 dyne/cm2 tions were calculated from the number of rolling PMN revealed that L-selectin-shedding was largely responsible in relation to total adhesion as a measure for adhesion for the decreased adhesion of activated PMN under nor- efficiency (Figure 4). For similar reasons mean rolling mal flow (Table 2). Blocking L-selectin decreased adhe- velocities were calculated (Figure 5) since rolling velocity sion of non-activated PMN by 30% (P < 0.05) down to is inversely correlated with the chance of a PMN to values obtained with activated PMN whereas no effect become adherent [27]. was observed on activated PMN. Blockade of P-selectin On maximally activated HUVEC with upregulated had no significant effect in both groups, suggesting that E-selectin, PMN-activation had no influence on rolling P-selectin played no role on intact HUVEC after four fraction (P = 0.59, Figure 4e). This indicated that L- hours LPS-activation. Consequently, only E-selectin selectin shedding decreased adhesion mainly by impairing remained functional under the condition of systemic initial capture under normal shear whereas E-selectin was hyper-inflammation and blocking the molecule in sufficient to translate existing rolling interactions into firm Ploppa et al. Critical Care 2010, 14:R201 Page 6 of 13 http://ccforum.com/content/14/6/R201

Figure 3 Interdependent effects of shear stress and cell activation on PMN adhesion. Adhesion of neutrophils under different activation protocols (mean ± SEM; n = 4), (a) non-activated controls, (b) activation with 100 ng/ml LPS and (c) activation with 10 ng/ml LPS. Blank symbols indicate activated PMN, filled symbols indicate non-activated PMN. (d-f) show the corresponding curves for predicted adhesion determined by ANCOVA of logarithms (continuous line: non-activated PMN, discontinuous line: activated PMN). Under all conditions of activation decreasing shear stress increased adhesion (P < 0.01; ANCOVA). On maximal activated endothelium activation of PMN decreased adhesion in comparison to non-activated PMN ((b and e), P < 0.01, ANCOVA). On sub-maximal activated endothelium (c and f), activation of PMN also decreased adhesion in comparison to non-activated controls, especially under conditions of low shear stress (P < 0.01, ANCOVA). adhesion. Accordingly, E-selectin maintained slow rolling On sub-maximally activated HUVEC without E- velocities above 0.5 dyne/cm2 whereas markedly higher selectin, co-activated PMN showed significantly velocities were observed on HUVEC lacking E-selectin increased rolling fractions at all levels of shear stress, (Figure 5). Because selectin function requires the presence indicating decreased adhesion efficiency (P <0.05,Fig- of shear-induced torque [36], rolling velocities increased ure 4f). Since HUVEC+/PMN- and HUVEC+/PMN+ sharply when reaching the shear-dependent threshold for differed in CD11b expression (Figure 2), the higher roll- E-selectin function. With further reduction in shear, rolling fraction at low shear stress indicated altered qualita- ing velocities then decreased along with the reduction in tive integrin activation despite numerical upregulation. flow velocity. Accordingly, rolling velocities in HUVEC+/PMN+ Ploppa et al. Critical Care 2010, 14:R201 Page 7 of 13 http://ccforum.com/content/14/6/R201

Table 2 Effects of PMN-activation on selectin function at 2 dyne/cm2 Adhesion [PMN/mm2] Blocking antibody HUVEC++/PMN- HUVEC++/PMN++ HUVEC++/PMN++/PLT++ NONE 1042 ± 61 591 ± 43 1313 ± 25 L- 744 ± 67 * 607 ± 56 ns vs NONE Ø P- 833 ± 59 ns vs NONE 596 ± 85 ns vs NONE 396 ± 35 * vs NONE E- 504 ± 55 * vs NONE 267 ± 32 * vs NONE Ø L-/P- 674 ± 48 ns vs L- ØØ E-/P- 504 ± 91 230 ± 12 ns vs E- Ø L-/E- 405 ± 59 * vs L- ØØ L-/E-/P- 343 ± 40 Ø Ø Adhesion in lipopolysaccharide-activated cultures (100 ng/ml; HUVEC++, PMN++, PLT++) at 2 dyne/cm2. Ø (not determined); * and ns (P < 0.05 versus indicated group or not significant, respectively). Statistical analysis with paired t-tests and correction after Bonferroni-Holm (mean ± SEM; n =4). For comparison, background adhesion in non-activated cultures (HUVEC-/PMN-) at 2 dyne/cm2 revealed 247 ± 52 PMN/mm2. HUVEC, human umbilical venous endothelial cells; PMN, polymorphonuclear neutrophils; PLT, platelets; L-, leukocyte selectin; P-, platelet selectin; E-, endothelial selectin; SEM, standard error of the mean. equalled those that have been reported for the low-affi- activation, adhesion molecule expression, shear stress nity configuration of b2-integrins [37]. and a platelet-covered endothelial injury on PMN- adhesion. Modulation of PMN-HUVEC interactions by adherent In order to mimic different stages of inflammation, as platelets they are frequently observed during the time course of To differentiate effects of endothelial activation from severe sepsis, various constellations of PMN and endothe- effects of endothelial injury on PMN recruitment lial activation were combined. Maximal activation of both [29-32,38] we examined the adhesion of activated PMN PMN and HUVEC was considered to reflect maximal sys- to platelet-covered endothelial lesions. temical inflammation in the hyper-inflammatory stage of The presence of platelets was the strongest variable for sepsis where high concentrations of circulating mediators adhesion of activated PMN. At all levels of shear stress induce activation of leukocyte and endothelial cell adhe- PMN adhesion on platelet-covered, injured HUVEC sion molecule expression systemically throughout the cir- increased significantly when compared to intact HUVEC culation [2,3,10]. Submaximal activation induced (P < 0.01, Figure 6). At 2 dyne/cm2 PMN adhesion upregulation of CD11b and downregulation of L-selectin increased 2.7-fold in maximally activated HUVEC++/PMN on PMN to the same degree as the maximal activation ++/PLT (Figure 6a, b). In sub-maximally activated HUVEC did, however, without having an effect on endothelial cell +/PMN+/PLT an even larger 10-fold increase in adhesion adhesion molecule expression. Since this pattern of was observed (Figure 6c, d). Additionally, platelets largely expression has been previously documented in studies on increased adhesion efficiency as documented by the consis- endotoxin tolerance and later hypo-inflammatory sepsis, tently lower rolling fractions at both LPS concentrations we used the sub-maximal LPS-activation as a model for and all levels of shear stress (P < 0.01, Figure 7). Accord- the hypo-inflammatory stage [2,3,10-17]. ingly, the rolling velocities remained low in both maximally Apart from the different stages of inflammation, adhe- and even sub-maximally activated co-cultures (4.5 ± sion molecule expression during systemic sepsis differs 1.0 μm/s and 5.8 ± 1.5 μm/s, respectively). from local inflammation in another important aspect. In Blockade of P-selectin revealed that the increased adhe- local inflammation upregulation of leukocyte integrins sion was largely due to platelet P-selectin. In contrast to and shedding of L-selectin does not occur before enter- its lacking effect in intact HUVEC++/PMN++, P-selectin ing the inflamed tissue [1]. To account for this differ- blocking WASP12.2 decreased PMN adhesion in injured ence, activated HUVEC were used in combination with HUVEC++/PMN++/PLT by 70% (P <0.01,Table2) non-activated PMN to mimic local inflammation below the values obtained in intact HUVEC++/PMN++. whereas PMN were treated with the same LPS concentrations as HUVEC to model sepsis-associated systemic Discussion inflammation. Toprovidemoreinsightintothemechanismsthat The results demonstrate that impaired recruitment of might explain the occurrence of disseminated leukocyte- systemically activated PMN to local sites of inflamma- related tissue damage in spite of an impaired leukocyte tion during severe sepsis [2,3,8-10] can be explained by recruitment to local sites of inflammation during severe two mechanisms. At normal shear stress, shedding of sepsis, we investigated the interdependent effects of cell L-selectin reduced adhesion in our experiments by Ploppa et al. Critical Care 2010, 14:R201 Page 8 of 13 http://ccforum.com/content/14/6/R201

Figure 4 Interdependent effects of shear stress and cell activation on PMN rolling. Rolling of neutrophils under different activation protocols (mean ± SEM; n = 4), (a) non-activated controls, (b) activation with 100 ng/ml LPS and (c) activation with 10 ng/ml LPS. Blank symbols indicate activated PMN, filled symbols indicate non-activated PMN. (d-f) show the corresponding curves for predicted rolling fractions determined by ANCOVA of logarithms (continuous line: non-activated PMN, discontinuous line: activated PMN). Rolling increased with decreasing shear stress in all cultures (a-c). On non-activated (d) and sub-maximal activated HUVEC (f) decreased shear stress increased the rolling fraction (P < 0.05, ANCOVA) whereas it had no effect under maximal LPS-activation (e). Activation of PMN induced higher rolling fractions in comparison to non-activated PMN at sub-maximal activation ((f), P < 0.05, ANCOVA). impairing initial capture. With reduction in shear CD11b remained upregulated on sub-maximally acti- stress this mechanism became less important and vated PMN, this finding indicates a dissociated quanti- adhesion increased. However, adhesion of activated tative and qualitative integrin-activation as the second PMN still appeared reduced in comparison to non- mechanism for altered adhesion of activated PMN. activated PMN. This reduction was most obvious in Integrin-dependent adhesion involves a cooperative the sub-maximally activated group at shear stresses and sequential process of LFA-1-dependent initiation where primary integrin-dependent adhesion occurs and Mac-1-dependent stabilization [39]. The increased independently of selectin interactions [35,36]. Since integrin-affinity, necessary to form bonds with their Ploppa et al. Critical Care 2010, 14:R201 Page 9 of 13 http://ccforum.com/content/14/6/R201

macrohemodynamics have been restored [23-25], this finding suggests that variations in shear stress largely influence leukocyte accumulation once systemic inflammation has evolved. Additionally, their influence seems to increase as soon as hyper-inflammation has turned into hypo-inflammation as might occur early, especially in those patients with poor prognosis [12,13]. Far exceeding the effects of shear stress is the platelet- covered endothelial lesion, which proved to be the strongest determinant of PMN-adhesion at all levels of shear stress. In maximally activated cultures, PLT-PMN interactions increased PMN adhesion by two-fold. At the sub- maximal LPS dose, an even more dramatic 10-fold increase was observed. Both findings indicate that endothelial cell damage gains a leading role for the spatial distribution of leukocyte accumulation through PLT- Figure 5 Effects of shear stress and different conditions of PMN interactions under conditions of systemic leukocyte activation on rolling velocities. Plots of mean rolling velocities of >25 PMN (mean ± SEM; n = 4). Circles indicate maximally activated activation and becomes exceedingly pronounced when HUVEC++ (LPS 100 ng/ml) with non-activated or activated PMN true endothelial cell activation is outweighed by endothe- (PMN- and PMN++ respectively). Triangles indicate sub-maximally lial cell damage, as might occur in the hypo-inflammatory activated HUVEC+ (LPS 10 ng/ml) with non-activated or activated stage of severe sepsis [11-17,30,32]. At sites of endothelial PMN (PMN- and PMN+ respectively). Square symbols indicate non- cell injury, platelet activation occurs through contact to activated controls (HUVEC-/PMN-). In non-activated and sub- maximal activated cultures without E-selectin expression, rolling the subendothelial matrix and does not become altered PMN were too few to calculate mean velocities above 1 dyne/cm2. when endothelial cell activation is impaired [34,38]. Pla- Maximal activation of HUVEC prevailed constant rolling velocities telet adhesion to the intact endothelium, in contrast, between 3 and 1 dyne/cm2 characteristic for selectin-interactions. requires the presence of endothelium-derived P-selectin Reduction of shear stress below a critical threshold increased rolling [34]. Although the latter mechanism contributes to leu- velocities followed by a decrease with further reduction of shear stress along with the reduction in hydrodynamic flow velocity. In kocyte accumulation in rodents, humans and primates cultures without E-selectin markedly increased rolling velocities were are not able to sustain endothelial P-selectin expression observed already at 1 dyne/cm2. beyond the very first minutes of inflammation because of a lack in transcriptional regulation [34,41]. Accordingly, blocking P-selectin had no effect on PMN-adhesion to endothelial ligands, is transient within minutes after intact HUVEC after four hours LPS-activation in our activation [40]. Accordingly, we observed decreased human adhesion experiments. integrin-dependent adhesion efficiency after PMN- Independent from endothelial cell activation platelet- activation and the rolling velocities equalled those that covered lesions provide a rich source of platelet-derived have been reported for the low affinity configuration of P-selectin [33,34]. In our experiments the high density of LFA-1 [37]. platelet- but not endothelium-derived P-selectin largely Reflecting the well-known inverse correlation of shear increased adhesion and adhesion efficiency as reflected by stress and adhesion in general [19-22] PMN-adhesion the different effect of P-selectin blockade on intact and was largely influenced by shear stress in all cultures. injured HUVEC. Even in rodents, who are able to sustain More importantly, the net effect of shear stress depended endothelial P-selectin expression for a longer time than on the inflammatory state of the interacting cell popula- humans [34,41], platelet but not endothelial P-selectin tions. Firm adhesion of non-activated PMN to maximally contributes to leukocyte-related organ dysfunction during activated HUVEC showed the smallest susceptibility to severe inflammation [42-44]. In contrast to a previous shear stress, which seems reasonable for targeting leuko- study that interpreted adhesion of leukocytes from septic cytes to a local site of inflammation independent of varia- individuals to a platelet surface as a general sign for tions in postcapillary blood flow. As soon as the PMN increased leukocyte adhesiveness during sepsis [45], we, were activated, loss of L-selectin rendered cell interac- therefore, considered PMN adhesion to the platelet- tions increasingly susceptible to shear stress. In sub- covered subendothelial matrix as a model for leukocyte maximally activated cultures, shear stress became the accumulation in the injured, rather than the activated, but prevailing determinant of PMN adhesion. Regarding intact microvasculature in a source of infection. the heavily decreased flow velocities that may arise in Since the effects of shear stress, tissue hypoxia, cell small vessels of the septic microcirculation even when activation and cell injury are hardly distinguishable from Ploppa et al. Critical Care 2010, 14:R201 Page 10 of 13 http://ccforum.com/content/14/6/R201

Figure 6 Effects of endothelial injury, platelet interactions and shear stress on PMN adhesion. Adhesion of activated PMN (mean ± SEM; n = 4) on an endothelial lesion covered with platelets (filled symbols) or intact endothelium (blank symbols) under maximal and sub-maximal activation. (a) activation with 100 ng/ml LPS (HUVEC++/PMN++/PLT vs. HUVEC++/PMN++), and (c) activation with 10 ng/ml LPS (HUVEC+/PMN +/PLT vs. HUVEC+/PMN+). (b and d) show the corresponding curves for predicted adhesion determined by ANCOVA of logarithms (continuous line: intact HUVEC, discontinuous line: injured HUVEC with platelets). The presence of platelets significantly increased adherence of PMN under all conditions of activation and shear, with the most pronounced effect on sub-maximally activated endothelium (P < 0.01; ANCOVA). each other during sepsis in vivo and, in part, are species- systemic inflammation. Although this model is artificial related, we decided to use a flow chamber to examine in many aspects, flow chamber experiments have proven their interplay in a human setting. Clearly, this simpli- valid for studying cell interactions in a number of stu- fied in vitro model has other inherent limitations since dies including direct comparison with leukocyte adhe- it neither includes true infection nor simulates all sion in animals [26,46]. Additionally, the experimental aspects of sepsis in an intact organism. For instance, we model resulted in adhesion molecule patterns as had to abstain from inducing true endotoxin tolerance they have been observed under different stages of sepsis- since this would have required prolonged cell culture associated systemic inflammation in vivo [2,3,10,12-17]. with inevitable confounding effects on adhesion molecule expression in an otherwise comparative experimen- Conclusions tal setting. Additionally, the use of cell suspensions In summary, our findings indicate a maldistribution of instead of whole blood influences rheological properties systemically activated leukocytes away from sites of local and the fixed diameter of the flow channel precludes inflammation with intact endothelium and normal blood effects of luminal narrowing that may arise in small ves- flow towards sites with compromised perfusion or sels during leukocyte adhesion.Apartfromdirectly endothelial cell injury. Because of L-selectin shedding favouring further adhesion, these effects may also influ- and altered integrin function, this maldistribution might ence cell interactions in vivo by decreasing blood flow occur even during the early hyper-inflammatory stage. It and oxygen transport. seems to become exceedingly pronounced, however, As a necessary simplification instead, we used different when endothelial LPS sensitivity is decreased, as might LPS-concentrations and standardized reproducible occur in patients with hypo-inflammatory cytokine pro- hydrodynamic conditions in an otherwise unchanged files [12-16]. From a clinical perspective, this suggests comparative model to investigate the mechanisms of that hemodynamic resuscitation should not only be tar- leukocyte accumulation during different stages of geted to increase oxygen delivery during the first hours Ploppa et al. Critical Care 2010, 14:R201 Page 11 of 13 http://ccforum.com/content/14/6/R201

Figure 7 Effects of endothelial injury, platelet interactions and shear stress on PMN rolling. Rolling of activated PMN (mean ± SEM; n =4) on an endothelial lesion covered with platelets (filled symbols) or intact endothelium (blank symbols) under maximal and sub-maximal activation. (a) activation with 100 ng/ml LPS (HUVEC++/PMN++/PLT vs. HUVEC++/PMN++), and (c) activation with 10 ng/ml LPS (HUVEC+/PMN +/PLT vs. HUVEC+/PMN+). (b and d) show the corresponding curves for predicted rolling-fractions determined by ANCOVA of logarithms (continuous line: intact HUVEC, discontinuous line: injured HUVEC with platelets). In contrast to intact HUVEC, rolling fractions on platelet-covered endothelial lesions remained low at both LPS concentrations and all levels of shear stress (P < 0.01, ANCOVA). of sepsis but to normalize microvascular blood flow • Together these mechanisms favor the maldistribu- velocity as an option to prevent disseminated leukocyte tion of leukocytes away from local sources of inflam- accumulation throughout the course of the syndrome. mation and towards areas with compromised flow Regarding the role of platelets, our observations add a and/or endothelial damage. further piece to the puzzle of platelet-neutrophil interactions during severe inflammation. In addition to those Abbreviations studies that have documented a contributory role for ANOVA: analysis of variance; ANCOVA: analysis of covariance; E-selectin: platelets in leukocyte-related tissue damage [42-44], our endothelial selectin; HUVEC: human umbilical venous endothelial cells; ICAM- results suggest that they might gain a leading role as 1: intercellular adhesion molecule-1; IL-10: interleukin-10; LFA-1: lymphocyte function antigen-1; LPS: lipopolysaccharide; L-selectin: leukocyte selectin; soon as endothelial damage outweighs endothelial acti- mAb: monoclonal antibody; MAC-1: macrophage antigen-1; MFI: median of vation. Tailoring the various forms of anti-platelet thera- fluorescence intensity; PLT: platelets; PMN: polymorhponuclear neutrophils; pies to sepsis stage and immune balance may therefore P-selectin: platelet selectin; SEM: standard error of the mean; TNF-a: tumor necrosis factor-a. represent a promising approach to increase their effec- tiveness in the future. Acknowledgments The authors thank Alice Mager and Christof Zanke, technical assistants, Department of Anaesthesiology and Intensive Care Medicine, University of Key messages Tuebingen, Germany, for their help with the adhesion assays, Martin Eichner, • Activation of leukocytes renders adhesion increas- Department of Medical Biometry, Eberhard-Karls University of Tuebingen, for ingly susceptible to shear stress. his statistical expertise, and Klaus E Unertl, Department Chair, Department of • Anaesthesiology and Intensive Care Medicine, University of Tuebingen, for Presence of a platelet-covered endothelial injury his generous support. overcomes this effect and seems to become the pre- Financial support: Supported in part by a grant of the fortuene- vailing factor for leukocyte accumulation under the programme to B. Nohé (project 777-0-0, Medical Faculty, University of Tuebingen). condition of systemic inflammation. Ploppa et al. Critical Care 2010, 14:R201 Page 12 of 13 http://ccforum.com/content/14/6/R201

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doi:10.1186/cc9322 Cite this article as: Ploppa et al.: Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury. Critical Care 2010 14:R201.

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